Drug-carrying proteins transported into cells by endocytosis can have marked pharmacologic effects, provided active drug is released inside cells. They are excellent tools, therefore, to study the intracellular processing of macromolecules. This project will compare drug conjugates using the same drug (methotrexate, MTX) but differing either in their carrier or in their linkage of drug to carrier. Two non-degradable drug carriers will be used, i.e., poly(D-lysine) internalized by non-specific endocytosis, and anti-TNP:MTX-TNP-poly(D-homocitrulline) immune complexes internalized by Fc-receptor-mediated endocytosis. The linkages can be tailored so as to cleave in three different cellular environments. They will include a triglycine linkage cleaved by lysosomal proteases, an acid-sensitive cis-aconityl linkage hydrolysed in acidic intracellular organelles, and a disulfide linkage cleaved wherever reducing conditions prevail along the endocytotic pathway. The conjugates will be used to characterize these three intracellular compartments, especially the third, about which least is known. The compartments will be identified by subcellular fractionation following exposure of cells to conjugates containing 3H-MTX and 14C-labeled carriers. Since released drugs will diffuse out of organelles, a decrease in the 3H/14C-ratio will identify the fractions in which cleavage is taking place. Factors known to influence these intracellular functions (inhibitor of proteases, lysosomotropic amines, precursors and inhibitors of glutathion synthesis) will be tested for their effect on both the intracellular distribution of 3H-MTX and the cytotoxicity of the conjugate. The same MTX-linkages will be inserted in two further carriers which follow different endocytotic pathways, namely transferrin, and a monoclonal antibody against the SSEA-1 tumor antigen. The fate of tumor antigen-antibody complexes is largely unknown and will be clarified by comparison with that of known ligands. Defects in endocytotic functions have been associated with important diseases (atherosclerosis, storage diseases). This research will provide new information on the intracellular processing of macromolecules in endocytosis. It will also yield basic information on mechanisms of drug release from macromolecular carriers, including carriers that are selectively targeted to tumor cells.